PROJECT SUMMARY: Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown etiology characterized by progressive lung fibrosis. It leads to death within 5 years unless patients undergo a lung transplant. Up to 34,000 cases of IPF are diagnosed in the United States each year. IPF lungs are characterized by increased accumulation of (myo)fibroblasts, activated fibroblasts with a contractile phenotype that are believed to be key effector cells leading to matrix deposition. Although the source of (myo)fibroblasts has not been definitively established, recent studies suggest that a significant portion of these cells arise from alveolar epithelial cells (AEC), through the process of epithelial-to-mesenchymal transition (EMT). Numerous observations point to a key role for AEC in the initiation and development of IPF. Alveolar epithelium consists of cuboidal type 2 (AEC2) cells and long thin type 1 (AEC1) cells, each with distinct morphologies, functions, and patterns of gene expression. AEC2 are a major source of pulmonary surfactants. They are believed to both self-renew and serve as progenitors for AEC1 for restoration of epithelial integrity following injury. In contrast, AEC1 mediate gas exchange and are believed to be terminally differentiated and unable to proliferate. AEC have been shown to undergo EMT in vivo in response to transforming growth factor beta (TGF-), giving rise to (myo)fibroblasts. AEC differentiation can be recapitulated in vitro; in the absence of added growth factors, purified AEC2 differentiate into AEC1-like cells. In contrast, when treated with TGF-, AEC2 undergo EMT, producing (myo)fibroblasts. This unique model system allows the temporal dissection of events that drive differentiation in the peripheral lung under conditions of health and disease. This proposal aims to identify genome-wide transcriptional and epigenetic changes of human AEC2 at distinct time points as the cells transition to AEC1- like cells or to (myo)fibroblasts. For comparison, primary AEC2 and AEC1 cells from normal lungs, and AEC2 and (myo)fibroblasts from the lungs of IPF patients will be examined. The Specific Aims are: 1) Transcriptome profiling of human AEC2 during the transition to AEC1-like cells or (myo)fibroblasts. Primary cells from healthy lung (AEC2 and AEC1) and IPF lung (AEC2 and (myo)fibroblasts) will be similarly analyzed. 2) Epigenomic profiling of the cells from Aim 1, using genome-wide approaches to assess DNA methylation patterns, histone modifications, polycomb group complex occupancy, chromosome remodeling complex occupancy, and selected transcription factor occupancy. 3) Integration of the data from Aims 1 and 2 to model the temporal and spatial relationships between the various epigenetic modifications and transcription of mRNAs and small non- coding RNAs. 4) Validation of a subset of newly identified expression patterns, epigenetic control networks and regulatory pathways using independent cultures of AEC and (myo)fibroblasts. Knowledge of the transcriptional and epigenetic programs driving AEC (mis)differentiation will provide insights into potential new avenues for lung restoration and will help develop strategies to treat and ultimately prevent diseases such as IPF.